Hemostatic and Ultrasound-Controlled Bactericidal Silk Fibroin Hydrogel via Integrating a Perfluorocarbon Nanoemulsion.

Excessive blood loss and infections are the prominent risks accounting for mortality and disability associated with acute wounds. Consequently, wound dressings should encompass adequate adhesive, hemostatic, and bactericidal attributes, yet their development remains challenging. This investigation presented the benefits of incorporating a perfluorocarbon nanoemulsion (PPP NE) into a silk-fibroin (SF)-based hydrogel. By stimulating the ß-sheet conformation of the SF chains, PPP NEs drastically shortened the gelation time while augmenting the elasticity, mechanical stability, and viscosity of the hydrogel. Furthermore, the integration of PPP NEs improved hemostatic competence by boosting the affinity between cells and biomacromolecules. It also endowed the hydrogel with ultrasound-controlled bactericidal ability through the inducement of inner cavitation by perfluorocarbon and reactive oxygen species (ROS) generated by the sonosensitizer protoporphyrin. Ultimately, we employed a laparotomy bleeding model and a Staphylococcus aureus-infected trauma wound to demonstrate the first-aid efficacy. Thus, our research suggested an emulsion-incorporating strategy for managing emergency wounds.

Infiltrating Perfluorocarbon Nanoemulsion and Sensitizing Ultrasound Cavitation to Eradicate Biofilms.

Developing strategies for the treatment of bacterial biofilms is challenging due to their complex and resilient structure, low permeability to therapeutics, and ability to protect resident pathogens. Herein, we demonstrate that a polylysine-stabilized perfluorocarbon nanoemulsion is favored for penetrating biofilms and sensitizing the cavitation effect of low-intensity ultrasound, resulting in the dispersal of extracellular polymeric substances and killing of the protected cells. Through experiments, we observed a complete penetration of the nanoemulsion in a 40 µm Pseudomonas aeruginosa biofilm and demonstrated that it was induced by the fluidic perfluorocarbon, possibly attributing to its low surface tension. Furthermore, we presented an almost complete antibiofilm effect with a low-intensity ultrasound (1 MHz, 0.75 W/cm2, 5 min) in diverse cases, including cultured biofilms, colonized urinary catheters, and chronic wounds. During the treatment process, the perfluorocarbon phase enhanced the number and imploding energy of ultrasound cavities, thoroughly divided the biofilm structure, prevented biofilm self-healing, and sterilized the resident pathogens. Thus, the penetration and sensitization of the nanoemulsion might serve as a facile and potent strategy for eradicating biofilms in various applications.

Nano-Emulsified Perfluorooctyl Bromide Can Infiltrate Gram-Negative Bacteria and Sensitize Them to Ultrasound.

Gram-negative (G-) bacterial infections remain one of the most urgent global health threats, because the distinctive envelope structure hinders the penetration of therapeutics. Here, we showed that a perfluorooctyl bromide nanoemulsion (PFOB NE) uniquely interacts with G- bacteria. After cell envelope attachment, the PFOB can infiltrate the cell and was diffused throughout. In this process, it impaired the membranes by disintegrating phospholipid molecules, enhancing the consequent ultrasonic cavitation to break the envelope. We identified through ultrasound that the NE had remarkable bactericidal effects against various antibiotic-resistant pathogens. Using in situ sterilization, this approach accelerated the recovery of bacteria-infected murine skin wounds. Thus, combining PFOB and ultrasound might be an alternative tool for conquering the growing threat of G- pathogens.

Ultrasmall Day-Night Photocatalysts of LnYVO4:Eu3+ (Ln = Nd, Tm, and Er) Nanoparticles.

Day-night photocatalysts that can persistently generate reactive oxygen species (ROS) after ceasing light attracted intensive attention in diverse fields. However, current strategies of combining a photocatalyst and an energy storage material can hardly fulfill the demands, especially in size. We herein present a one-phase sub-5 nm day-night photocatalyst via simply doping Nd, Tm, or Er into YVO4:Eu3+ nanoparticles, efficiently producing ROS in both day and night modes. We demonstrate that the rare earth ions acted as a ROS generator, and Eu3+ and defects contributed to the long persistency. Furthermore, the ultrasmall size led to remarkable bacterial uptake and bactericidal efficacy. Our finding suggests an alternative mechanism of day-night photocatalysts that could be ultrasmall and thus may shed light on disinfection and other applications.

Cat scratch disease with generalized bone lesions in an immunocompetent child.

A 9-year-old immunocompetent girl with prolonged fever for 1 month was suspected of having a malignancy because of generalized bone abnormalities identified by MRI. Histopathology of liver tissues indicated the diagnosis of cat-scratch disease (CSD). Results of NGS, immunofluorescence and immunochemical assay confirmed the causative agent was Bartonella henselae. Paediatricians should increase their awareness of CSD as a cause for bone lesions, except for malignancy.

Bartonella type IV secretion effector BepC induces stress fiber formation through activation of GEF-H1.

Bartonella T4SS effector BepC was reported to mediate internalization of big Bartonella aggregates into host cells by modulating F-actin polymerization. After that, BepC was indicated to induce host cell fragmentation, an interesting cell phenotype that is characterized by failure of rear-end retraction during cell migration, and subsequent dragging and fragmentation of cells. Here, we found that expression of BepC resulted in significant stress fiber formation and contractile cell morphology, which depended on combination of the N-terminus FIC (filamentation induced by c-AMP) domain and C-terminus BID (Bartonella intracellular delivery) domain of BepC. The FIC domain played a key role in BepC-induced stress fiber formation and cell fragmentation because deletion of FIC signature motif or mutation of two conserved amino acid residues abolished BepC-induced cell fragmentation. Immunoprecipitation confirmed the interaction of BepC with GEF-H1 (a microtubule-associated RhoA guanosine exchange factor), and siRNA-mediated depletion of GEF-H1 prevented BepC-induced stress fiber formation. Interaction with BepC caused the dissociation of GEF-H1 from microtubules and activation of RhoA to induce formation of stress fibers. The ROCK (Rho-associated protein kinase) inhibitor Y27632 completely blocked BepC effects on stress fiber formation and cell contractility. Moreover, stress fiber formation by BepC increased the stability of focal adhesions, which consequently impeded rear-edge detachment. Overall, our study revealed that BepC-induced stress fiber formation was achieved through the GEF-H1/RhoA/ROCK pathway.

Bartonella kosoyi sp. nov. and Bartonella krasnovii sp. nov., two novel species closely related to the zoonotic Bartonella elizabethae, isolated from black rats and wild desert rodent-fleas.

The genus Bartonella (Family: Bartonellaceae; Order: Rhizobiales; Class: Alphaproteobacteria) comprises facultative intracellular Gram-negative, haemotropic, slow-growing, vector-borne bacteria. Wild rodents and their fleas harbor a great diversity of species and strains of the genus Bartonella, including several zoonotic ones. This genetic diversity coupled with a fastidious nature of the organism results in a taxonomic challenge that has led to a massive collection of uncharacterized strains. Here, we report the genomic and phenotypic characterization of two strains, members of the genus Bartonella (namely Tel Aviv and OE 1-1), isolated from Rattus rattus rats and Synosternus cleopatrae fleas, respectively. Scanning electron microscopy revealed rod-shaped bacteria with polar pili, lengths ranging from 1.0 to 2.0 µm and widths ranging from 0.3 to 0.6 µm. OE 1-1 and Tel Aviv strains contained one single chromosome of 2.16 and 2.23 Mbp and one plasmid of 29.0 and 41.5 Kbp, with average DNA G+C contents of 38.16 and 38.47 mol%, respectively. These strains presented an average nucleotide identity (ANI) of 89.9 %. Bartonella elizabethae was found to be the closest phylogenetic relative of both strains (ANI=90.9-93.6 %). The major fatty acids identified in both strains were C18:1ω7c, C18 : 0 and C16 : 0. They differ from B. elizabethae in their C17 : 0 and C15 : 0 compositions. Both strains are strictly capnophilic and their biochemical profiles resembled those of species of the genus Bartonella with validly published names, whereas differences in arylamidase activities partially assisted in their speciation. Genomic and phenotypic differences demonstrate that OE 1-1 and Tel Aviv strains represent novel individual species, closely related to B. elizabethae, for which we propose the names Bartonella kosoyi sp. nov. and Bartonella krasnovii sp. nov.

Protective effects of rapamycin induced autophagy on CLP septic mice.

Sepsis is a life-threatening condition that may develop to multiple organ failure and septic shock. Autophagy is considered to play an important role in the regulation of inflammation. The present study aims to investigate the protective role of mTORC1 inhibitor, rapamycin, on septic death using cecal ligation and puncture (CLP) mice model. Here, results showed that pretreatment with rapamycin reduced the pyroptosis of peritoneal macrophages stimulated by cecal contents and the release of inflammatory factors such as interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α); In septic mice, rapamycin treatment decreased the activation of inflammasome in lung, and alleviated the pathological injuries in lung, liver and spleen tissues during acute stage of sepsis. Treatment of rapamycin rescued animals from septic death significantly. Our results indicated that activation of autophagy is a potential strategy to regulate the excessive inflammation in acute stage of sepsis.

Bartonella quintana type IV secretion effector BepE-induced selective autophagy by conjugation with K63 polyubiquitin chain.

Bartonella effector proteins (named Beps) are substrates of VirB type IV secretion system for translocation into host cells evolved in Bartonella spp. Among these, BepE has been shown to protect cells from fragmentation effects triggered by other Beps and to promote in vivo dissemination of bacteria from the dermal site of inoculation to the bloodstream. Bacterial pathogens secreted effectors to modulate the interplay with host autophagy, either to combat autophagy to escape its bactericidal effect or to exploit autophagy to benefit intracellular replication. Here, we reported a distinct phenotype that selective autophagy in host cells is activated as a countermeasure, to attack BepE via conjugation with K63 polyubiquitin chain on BepE. We found that ectopic expression of Bartonella quintana BepE specifically induced punctate structures that colocalised with an autophagy marker (LC3-II) in host cells, in addition to filopodia and membrane ruffle formation. Two tandemly arranged Bartonella Intracellular Delivery (BID) domains in the BepE C-terminus, where ubiquitination of sister pairs of lysine residues was confirmed, were essential to activate host cell autophagy. Multiple polyubiquitin chain linkages of K27, K29, K33, and K63 were found to be conjugated at sites of K222 and K365 on BepE, of which K63 polyubiquitination on BepE K365 determined the selective autophagy (p62/SQSTM1 positive autophagy) independent of the PI3K pathway. Colocalisation of BepE with LAMP1 confirmed the maturation of BepE-induced autophagosomes in which BepE were targeted for degradation. Moreover, host cells employed selective autophagy to counter-attack BepE to rescue cells from BepE-induced endocytosis deficiency.

Lymphatic Circulation Disseminates Bartonella Infection Into Bloodstream.

The hallmark of Bartonella infection is long-lasting intraerythrocytic parasitism. However, the process of Bartonella bacteremia is still enigmatic. In the current study, we used Bartonella tribocorum to determine how Bartonella invasion into the bloodstream from dermal inoculation might occur. Bartonella was poorly phagocytized by peritoneal macrophages in vitro. Intracellular Bartonella survived and replicated in macrophages at an early stage of infection. Intracellular Bartonella inhibited spontaneous cell death of macrophages. They also inhibited Salmonella-induced pyroptosis and mildly reduced inflammasome activation through an unidentified mechanism. A rat model confirmed that Bartonella was also inadequately phagocytized in vivo, because numerous free-floating bacilli were observed in lymph collected from thoracic duct drainage as early as 2 hours after inoculation. Lymphatic fluid drainage in the bloodstream significantly reduced the bacterial load in the bloodstream. These findings illustrated a potential route by which Bartonella invade bloodstream from dermal inoculation before they are competent to infect erythrocytes.

Effects of Ketamine on Metabolomics of Serum and Urine in Cynomolgus Macaques (Macaca fascicularis).

In this study, a metabolomics approach based on nuclear magnetic resonance spectroscopy and pertinent multivariate data analyses was used to evaluate the effect of ketamine on metabolic markers in cynomolgus macaques. Principal component analysis and orthogonal projection to latent structure with discriminant analysis showed that ketamine (10 mg/kg) induced metabolic perturbations. Compared with the control group, ketamine-treated macaques had lower serum levels of α-glucose, myoinositol, lactate and succinate and lower urine levels of pyruvate and lactate. In contrast, the levels of leucine in serum and arginine in urine were significantly higher in the ketamine group. Our results also demonstrated that a single injection of ketamine influenced the major energy and amino acid metabolic pathways in cynomolgus macaques. Our study suggests that these influences should be considered in the design of experiments and the interpretation related blood and urine data from ketamine-sedated cynomolgus macaques.

New serotypes of porcine teschovirus identified in Shanghai, China.

Teschoviruses are widely endemic and commonly found in pig fecal samples. In this study, we collected fecal specimens from various pig herds and genotyped them based on the VP1 gene. Of 322 samples, 276 were positive, giving a PTV infectivity rate of 85.7 %. PTV4 was the most common serotype found in Shanghai, followed by PTV8 and PTV10. Interestingly, Some Shanghai strains belonging to a new PTV serotype were also isolated. In phylogenetic analysis, PTV SH8 did not correspond to any known serotype. PTV4 and PTV6 showed similar levels of sequence identity to PTV SH8. These data suggest that PTV SH8 is a new serotype, distinct from the new serotype PTV wild boar/WB2C-TV/2011/HUN, which clusters with PTV SH2, SH10, and SH25.

[Experimental study of the monitoring bias of pressure in intubation balloon using handheld pressure gauge].

OBJECTIVE: To explore the bias between the real pressure and the measured values when handheld pressure gauge (HPG) was used to monitor intermittently the pressure in the intubation balloon, so as to provide some measures for the correct use of HPG. METHODS: In the first part of the study, HPG was used to measure the pressure with the balloon connected with a three-way tube with which to control the inflation and deflation in a laboratory to measure the pressure in the air bag. After gaining the deviation in this in vitro experiment, it was tested and verified in vivo in adult patients undergoing endotracheal intubation. RESULTS: After 132 times of measurements, it was found that measurement with a HPG might result in an "inherent loss" (3.928±0.291) cmH2O (1 cmH2O=0.098 kPa, t=155.273, P=0.000) between inflation value [(30.000±0.000) cmH2O] and measured value [(26.072±0.291) cmH2O]. In addition, after 214 times repeated measurements, the pressure "loss" during disconnection of the gauge was as high as (1.196±0.954) cmH2O (t=18.348, P=0.000) between filled values [(30.000±0.000) cmH2O] and measured values [(28.804±0.954) cmH2O] and it was named as "error loss". At last, the total error was verified by clinical test, and it was (5.270±2.583) cmH2O (t=29.632, P=0.000) between pressure of filled value [(30.000±0.000) cmH2O] and measured value [(24.730±2.583) cmH2O]. CONCLUSIONS: When the balloon pressure was monitored intermittently with HPG, the real value should be the measured value plus the "error". In addition, subglottic aspiration should be done before the connection of the balloon to the gauge to prevent the secretions on the cuff falls into the deeper airway, and to maintain the cuff pressure at 30 cmH2O.

Molecular detection of genogroup I and II picobirnaviruses in pigs in China.

In this study, a total of 187 stool specimens were collected from a pig farm in Hunan province of China, from November 2011 to June 2012. 39 (20.9 %) stool specimens were positive for picobirnaviruses using reverse transcription-polymerase chain reaction. Among 39 stool specimens, 84.6 % (33/39) were identified to be genogroup I (prototype 1-CHN-97), 38.5 % (15/39) belonged to genogroup II (prototype 4-GA-91), and 23.1 % (9/39) of which showed the evidence of genogroup I picobirnavirus were also positive for genogroup II picobirnaviruses. Picobirnaviruses exist in pigs which were divided into five groups according to the age and physiological status. Nineteen representative strains of genogroup I picobirnaviruses and eleven strains of genogroup II picobirnaviruses detected in this study were selected to analyze their phylogenetic relationships with other picobirnaviruses reference strains. The phylogenetic tree analysis suggested the prevalence of multiple picobirnaviruses in pigs in China.

HEV-ORF3 Encoding Phosphoprotein Interacts With Hepsin.

BACKGROUND: Hepatitis E virus (HEV) is a major causative agent of acute clinical hepatitis in adults through much of Asia, the Middle East and Africa. Open reading frame 3 (ORF3) encodes around 120 amino acids of phosphorylation protein that associates with the cytoskeleton, while its precise biological function is still unknown. OBJECTIVES: In order to understand the function of ORF3 protein (pORF3) in depth, HEV ORF3 interacting proteins were screened in human hepatocytes cDNA library using two-hybrid system techniques and further verification of the interactions were carried out through co-immunoprecipitation (Co-IP). MATERIALS AND METHODS: The Cyto-Trap two-hybrid system technology, a classical method for analyzing protein interactions, was used to screen the pORF3 interacting proteins from human hepatocytes cDNA library. RESULTS: Through the Cyto-Trap two-hybrid system, eight proteins interacting with pORF3 were winnowed. The Co-IP results confirmed that hepsin which is reported to function as the inhibitor of several tumors reacted with pORF3. CONCLUSIONS: Out of eight screened proteins interacting with pORF3, hepsin was confirmed to have specific interactions with pORF3.

Rapid and real-time detection of Porcine Sapelovirus by reverse transcription loop-mediated isothermal amplification assay.

The present study describes the development and validation of a one-step, single-tube, and real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) detecting Porcine Sapelovirus. RT-LAMP characterized by one strand displacement reaction with the specific stem-loop structure and Bst DNA polymerase could be finished in 60 min under isothermal condition at 63 °C. RT-LAMP assay showed higher sensitivity with 10(1) copies/µL than RT-PCR for the detection of Sapelovirus. The specificity of RT-LAMP assay was validated by the absence of any cross-reaction with other closely related virus in Picornaviridae group and other common virus causing porcine diarrhea. 7 positive Sapelovirus infection out of 63 fecal samples were identified using RT-LAMP, while 5 positive samples were determined by a conventional RT-PCR. A cost-effective method for Saplovirus detection with high sensitivity and specificity was developed and evaluated.

Depolymerization of cytokeratin intermediate filaments facilitates intracellular infection of HeLa cells by Bartonella henselae.

Bartonella henselae is capable of invading epithelial and endothelial cells by modulating the function of actin-dependent cytoskeleton proteins. Although understanding of the pathogenesis has been increased by the development of an in vitro infection model involving endothelial cells, little is known about the mechanism of interaction between B. henselae and epithelial cells. This study aims to identify the binding candidates of B. henselae in epithelial cells and explores their effect on B. henselae infection. Pull-down assays and mass spectrometry analysis confirmed that some of the binding proteins (keratin 14, keratin 6, and F-actin) are cytoskeleton associated. B. henselae infection significantly induces the expression of the cytokeratin genes. Chemical disruption of the keratin network by using ethylene glycol tetraacetic acid promotes the intracellular persistence of B. henselae in HeLa cells. However, cytochalasin B and phalloidin treatment inhibits B. henselae invasion. Immunofluorescent staining demonstrates that B. henselae infection induces an F-actin-dependent rearrangement of the cytoskeleton. However, we demonstrated via immunofluorescent staining and whole-mount cell electron microscopy that keratin intermediate filaments are depolymerized by B. henselae. The results indicate that B. henselae achieves an intracellular persistence in epithelial cells through the depolymerization of cytokeratin intermediate filaments that are protective against B. henselae invasion.

Complete genome of transmissible gastroenteritis virus AYU strain isolated in Shanghai, China.

Transmissible gastroenteritis virus strain AYU was isolated in Shanghai. The complete genome has a length of 28,582 bp and contains seven open reading frames. Sequence analysis suggested that Shanghai strain AYU and U.S. strain Purdue P115 are derived from a common ancestor, as they have 99.6% similarity at the nucleotide level.

Staining of Bartonella henselae with carboxyfluorescein diacetate succinimidyl ester for tracking infection in erythrocytes and epithelial cells.

Bartonella infection (Bartonella henselae in particular) is responsible for a widening spectrum of human diseases. The persistent colonization of erythrocytes is a feature of Bartonella infection. Endothelial and epithelial cells are also widely used to study the pathogenesis of bartonellosis in vitro. Exploring a convenient method for visualizing the bacillus without affecting infectivity would be very interesting. Carboxyfluorescein diacetate succinimidyl ester (CFSE) has been previously used for staining several bacterial species to study their adhesion to host cells. The present study demonstrated the efficiency and safety of using CFSE in staining B. henselae. The staining of bacillus-invaded erythrocytes and epithelial cells in vitro successfully allowed for flow cytometry and confocol microscopy analyses. Parallel tests using untreated bacteria confirmed that CFSE staining did not result in side effects on the infectivity of B. henselae. Labeling Bartonella with CFSE is a valuable method for studying the bacteria-host interaction.

Molecular detection of Torque teno mini virus (TTMV) in China.

In this study, 176 serum specimens were collected from hospitalized cardiovascular diseases patients in Shanghai, China. 140 samples (79.5%) were positive with the primers located in non-coding region, and 30 samples (17.04%) were identified to be positive with group-specific primers. Of the 30 samples, 9 (5.11%) were classified to group 1, 23 (13.07%) were clustered to group 2, and 3 belonged to group 3. Five samples were confirmed to be dual infection with different groups of TTMV, and no sample was found to be infected with groups 4-6.